1. Field of the Invention
The present invention relates to the synthesis of biologically important .alpha.-glycosides of N-acetylgalactosamine, and provides versatile new intermediates for an improved method of preparing oligosaccharides, glycosylamino acids, and glycopeptides which are useful in treating and diagnosing cancers as well as other disease states.
2. Description of the Background Art
Mucins are glycoproteins found in saliva, gastric juices, and the like that form viscous solutions and act as lubricants or protectants on external and internal body surfaces (Florey, et al., Br. J. Exp. Pathol., 13: 269, 1932; Gottschalk, et al., Glycoproteins: Their Composition, Structure and Function, Gottschalk, A., Ed., Elsevier, New York, 1972; Gottschalk, et al., Biochem. Biophys. Acta, 54: 226, 1961; Hill, et al., J. Biol. Chem., 252: 3791, 1977; Roussel, et al., Biochimie, 70: 1471, 1988). Mucins are of typical high molecular weight compounds, often greater than 100,000 Daltons, and are extensively glycosylated (up to 80% glycan).
Mucin glycans (Podolsky, J. Biol. Chem., 260: 8262, 1985) are bound to the "core" protein (or apaproteini) of the mucin through oxygen atoms in the side chains of serine and/or threonine residues. This is termed as an O-linkage. The reducing sugar of the glycan (the sugar directly attached to the core protein) is usually N-acetylgalactosamine (Stryer, Biochemistry, 3rd Edition, W. H. Freeman & Co., New York, 1988, p. 298). The main sugar constituents of the mucin glycans are galactose, N-acetylgalactosamine, N-acetylglucosamine, fucose, and sialic acid (Filipe, Invest. Cell, Pathol., 2: 195, 1979).
Mucins are normally found in the secretions of many epithelial glands. However, they can also occur cell-bound as integral membrane proteins (Simmons, et al., J. Immunol., 148: 267, 1992; Marchesi, et al., Ann. Rev. Biochem., 45: 667, 1976; Carraway, et al., Mol. Cell. Biochem., 72: 109, 1986).
Cancer-associated mucins are produced by tumor tissue of epithelial cell origin (Burcheil, et al., Cancer Res., 47: 5476, 1987; Jerome, et al., Cancer Res., 51: 2908, 1991). A major difference between the normal mucins and the aberrant, cancer-associated mucins, is in the structures of their respective glycans. The extent of glycosylation of cancer associated mucins is lower than that of their normal counterparts (Hull, et al., Cancer Commun., 1:261, 1989). For example, while the normal mucin associated with human milk fat globules contains primarily the tetrasaccharide glycan, .beta.Gal 1-4 .beta.GlcNAc 1-6(.beta.Gal 1-3)GalNAc and its sialylated analogs, the GlcNAc 1-6 transferase seems to be defective in breast cancer cells (Hull, et al., vide supra). As a result, the non reducing .beta.1-4-linked galactosyl residue is also absent and the glycans of breast cancer-associated mucins are characteristically incomplete. Typical glycan structures found in cancer-associated mucins are the solitary N-acetylgalactosaminyl residue (Tn-determinant) or the disaccharide .beta.Gal 1-3 .alpha.GalNAc (T-determinant; Springer, Science, 224: 1198, 1984). In both the Tn and TF determinants, additional 2-6-linked .alpha.-sialyl groups may be attached to the N-acetylgalactosaminyl moiety, resulting in the sialyl-Tn (STn) and sialyl-2-6T determinants (Hanish, et al., Biol. Chem. HoppeSeyler, 370: 21, 1989; Hakomori, Adv. Cancer Res., 52: 257, 1989; Oristoft, et al., Int. J. Cancer, 45: 666, 1980; Samuel, et al., Cancer Res., 50: 4801, 1990).
More than 90% of primary carcinomas and their metastases contain the Tn and T determinants in immunoreactive form on their external surface membranes (Springer, vide suora). The altered glycan determinants displayed by the cancer-associated mucins are recognized as non-self or foreign by the patient's immune system, resulting in autoimmune responses.
The presence of Tn, TF, STn and sialyl 2-6TF-antigens on cancer cells and the autoimmune response to these structures have been used for diagnostic and therapeutic procedures. As cancer markers, Tn and TF permit early immunohistochemical detection and prognostication of the invasiveness of some carcinomas (Springer, vide supra). The presence of the sialyl-Tn hapten on tumor tissue has been identified as an unfavorable prognostic parameter (Itzkowitz, et al., Cancer, 66: 1960, 1990; Yonezawa, et al., Am. J. Clin. Pathol., 98: 167, 1992).
Measurements of the autoimmune response directed against the Tn and TF determinants permit detection of carcinomas with greater sensitivity and specificity, earlier than has previously been possible. Also, the extent of expression of Tn and TF determinants often correlates with the degree of differentiation of carcinomas (Springer, vide supra).
The nature of the Tn, TF and STn structures as tumor-associated antigens has led to their use in active specific immunotherapy of cancers. Specifically, the Tn, TF and STn determinants have been chemically synthesized and attached to carrier proteins to form artificial antigens; these were administered to cancer patients in combination with cyclophosphamide and the Ribi DETOX.RTM. adjuvant (MacLean, et al., J. Immunother., 11: 292, 1992; MacLean, et al., Cancer Immunoth., 36: 215, 1993).
Synthesis of glycosylated amino acids has become an important field of carbohydrate chemistry. More recently, synthesis of glycosylated segments of mucins and glycoproteins that are associated with tumors, pathogens and viruses have become extremely important because of their ability to function as immunogens. These synthetic antigens are chemically well-defined and are easily purified. However, a major concern in any synthetic vaccine development is large scale manufacturing for commercial purposes. The key factors to successful large scale production are commercial availability of the raw materials, the number of synthetic steps, quantity and the types of by-products formed and overall yields.
Glycosides of the Tn and TF haptens, and of their sialylated analogs, have been synthesized and conjugated to proteins or synthetic peptide carriers for use in diagnostic and therapeutic applications (Lemieux, et al., U.S. Pat. Nos. 4,794,176; 4,308,376; 4,362,720 and 4,195,174; Treman, U.S. Pat. No. 4,866,045 Can. J. Chem., 57: 1244, 1979). Alternatively, the tumor-associated carbohydrate antigens have been isolated from natural sources and used in immunotherapeutic applications (Kjeldsen, et al., International Patent Application PCT/US89/00966, published as W0 89/08711 Sep. 21, 1989). The synthetic glycosides used have generally involved an aglycon moiety from which reactive functionality, suitable for coupling, could be generated without altering the saccharide portion of the hapten glycoside. The `activated` hapten glycosides were then reacted with amino groups of the proteins or peptide carriers to form an amide or a Schiff base linkage which is further reduced to an amine. However, the artificial antigens prepared in this manner do not comprise the natural carbohydrate-peptide linkages, and therefore they induce a response against the epitopes contained in both the saccharide as well as synthetic aglycon. To overcome this limitation, glycosyl amino acids have been synthesized by linking the Tn or TF haptens to protected serine or threonine derivatives; these were then assembled into glycosyl oligopeptides involving two, three or four amino acid residues (Paulsen, et al., Carbohydr. Res., 109: 89, 1982; Bencomo, et al., Carbohydr. Res., 116: C9, 1983; Iijima, et al., Carbohydr. Res., 186: 95, 1989).
For the synthesis of the required .alpha.-N-acetylgalactosaminides of hydroxyamino acids, glycosyl donors directly derived from N-acetylgalactosamine are unsuitable because the acetamido group directs glycoside formation toward the .beta.-anomeric configuration through the neighboring group participation, and gives rise to formation of side products such as oxazolines (1c). Indeed, direct use of N-acetylgalactosamine has only been described under the conditions of the Fischer glycosidation (Flowers, et al., J. Org. Chem., 30: 2041, 1965). However, the Fisher glycosidation is limited to aglycons derived from aliphatic and aromatic primary alcohols, while hydroxy amino acids such as serine and threonine are unsuitable due to their instability at the reaction conditions.
To obtain practical quantities of .alpha.-N-acetylgalactosaminides of hydroxyamino acids, the art has heretofore relied on a circuitous route using 2-azido-2-deoxygalactose derivatives as glycosyl donors and precursors of the N-acetylgalactosaminyl residues. As distinct from the acetamido group, the 2-azido group of the corresponding glycosyl donors does not participate in the reactions at the anomeric center resulting in the formation of side products, such as oxazolidines. Following the glycosidation step, the 2-azido-2-deoxyglycosides must be reduced to the amino glycoside and N-acetylated. By this indirect technique, .alpha.-N-acetylgalactosaminides of hydroxy amino acids may be obtained (Paulsen, et al., Bencomo, et al., and Iijima et al, vide supra). The required 2-azido-2-deoxy-galactosyl halide donors may be obtained via azidonitration of D-galactal, according to the process of Lemieux in U.S. patents cited above, Can. J. Chem., 57: 1244, 1979; or according to Paulsen, et al., (Chem. Ber. 111, 2358, 1978), starting from 1,6;2,3-dianhydro-D-talcpyranose. 2-Azido-2-deoxygalactopyranosyl halide intermediates may also be directly prepared by azidochlorination of D-galactal derivatives, as shown by Naicker, et al., (U.S. Pat. No. 4,935,503). All of these processes require relatively expensive starting materials, add several steps to the already lengthy syntheses, and are poorly adaptable to commercial-scale manufacturing of these important synthetic antigens. Therefore, a need exists for a shorter, more practical process to prepare glycosylamino acids in commercial quantities.
Earlier attempts to make .alpha.-glycosides of N-acetylgalactosamine with hydroxy amino acids involved the coupling of protected N-acetylgalactosamine donors with protected serine or threonine compounds under Koenigs-Knorr, using silver salt, or Helferich, using a mercuric salt as a catalyst. Both the Koenigs-Knorr and Helferich syntheses yield solely or predominantly .beta.-glycosides (1,2-trans) when a participating group is present.
Ferrari and Pavia, in Carbohydr. Res. 79: c1-c7, 1980; Biorg. Chem., 11: 82-85, 1982, reacted 3,4,6-triacetyl, 2-azido, D-galactosyl chloride, with a C.sub.2 non-participating group, with serine or threonine derivatives in the presence of mercuric cyanide to give the corresponding .alpha.-glycosylamino acid in 66 and 45% yield, respectively. This method involves the laborious and circuitous synthesis of a 2-azido galactosyl donor prior to glycosidation. Paulsen and Holck, reporting in Carbohydr. Res., 109: 89-108, condensed the same chloride with a serine derivative in the presence of silver carbonate, Drierire.RTM., 4A molecular sieves and silver perchlorate in toluene/dichloromethane to give an 85% yield of glycoamino acid, the ratio of .alpha. to .beta. being 19:1. The azido group in the glycosides can be converted to acetamido group by reduction to amino group and N-acetylation. The required conditions for reduction to the amino group further complicates the glycopeptide synthesis by removing the Fmoc group on the amino acid, which may also acetylate the amino acid rendering it unsuitable for glycopeptide synthesis.
Attempts to make use of trichloroacetimidate of 3,4,6 triacetyl N-acetylgalactosamine (Gandhi, et al., XV Intnl. Carb. Symp. Aug. 12-17, Yokohama, Japan, 1990) as a donor resulted mostly in the formation of oxazoline (1c) and a very poor yield of .alpha.-glycoside (.alpha. to .beta. ratio of 1:5), which proved not to be commercially viable.